Rapid Biodegradation of the Herbicide 2,4-Dichlorophenoxyacetic

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Rapid biodegradation of the herbicide 2,4dichlorophenoxyacetic acid by Cupriavidus gilardii T-1 Xiangwei Wu, Wenbo Wang, Junwei Liu, Dandan Pan, Xiaohui Tu, Pei Lv, Yi Wang, Haiqun Cao, Yawen Wang, and Rimao Hua J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 24 Apr 2017 Downloaded from http://pubs.acs.org on April 24, 2017

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Journal of Agricultural and Food Chemistry

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Rapid biodegradation of the herbicide 2,4-dichlorophenoxyacetic acid by

2

Cupriavidus gilardii T-1

3 4

Xiangwei Wu a, WenboWang a, Junwei Liu a, Dandan Pan a, Xiaohui Tu a, Pei Lv a, Yi Wang a,

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Haiqun Cao a, Yawen Wang a, Rimao Hua a, *

6 7 8

a

College of Resources and Environment, Anhui Agricultural University, Key Laboratory of Agri-food Safety of Anhui Province, Hefei 230036, P. R. China

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*Corresponding author: Rimao Hua

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Tel/Fax: +86-551-65786296

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E-mail: [email protected]; [email protected]

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ACS Paragon Plus Environment


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Abstract:

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Phytotoxicity and environmental pollution of residual herbicides have caused much public

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concern during the past several decades. An indigenous bacterial strain capable of degrading

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2,4-dichlorophenoxyacetic acid (2,4-D), designated T-1, was isolated from soybean field soil and

27

identified as Cupriavidus gilardii. The strain T-1 degraded 2,4-D 3.39 times faster than the model

28

strain

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2-methyl-4-chloro-phenoxyacetic

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2-(2,4-dicholrophenoxy)-propionic acid (2,4-DP). Suitable conditions for 2,4-D degradation were

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pH 7.0-9.0, 37-42 °C and 4.0 mL of inoculums. Degradation of 2, 4-D was

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concentration-dependent. 2,4-D was degraded to 2,4-dichlorophenol (2,4-DCP) by cleavage of

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ether bond, then to 3,5-dichlorocatechol (3,5-DCC) via hydroxylation, and followed by

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ortho-cleavage to cis-2-dichlorodiene lactone (CDL). The metabolites 2,4-DCP or 3,5-DCC at 10

35

mg L-1 were completely degraded within 16 h. Fast degradation of 2,4-D and its analogues

36

highlights the potential for use of C. gilardii T-1 in bioremediation of phenoxyalkanoic acid

37

herbicides.

Cupriavidus

necator

JMP134. acid

T-1 (MCPA),

could MCPA

also

efficiently isooctyl

ester,

degrade and

38 39 40

Key words: 2,4-D; Biodegradation; Cupriavidus gilardii; Phenoxyalkanoic acid herbicide; degradation pathway; Degradation rate.

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2


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1 Introduction

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2,4-dichlorophenoxyacetic acid (2,4-D), a phenoxyalkanoic acid (PAA) herbicide, was first

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synthesized in 1941, and commercially marketed in USA in 1944.1 2,4-D has been widely and

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intensively used for controlling some annual and perennial broad-leaved weeds in wheat, corn,

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cotton, soybean, and tobacco corps and nonagricultural soils.2-4 However, this widespread and

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frequent application of 2,4-D might cause several toxicological, environmental contamination,

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human health problems5 and give rise to the emergence of herbicide-resistant weeds.6,7 Despite its

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short half-life in soils or aquatic environment, 2,4-D has been frequently detected in soil,

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groundwater and surface water at concentrations ranging from several micrograms per liter to

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hundreds of micrograms per liter.8-11

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A variety of toxic effects caused by 2,4-D have been reported. Previous studies have

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suggested that 2,4-D might influence the physiological and behavioral consequence of aquatic

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organisms12 and be associated with gastric cancer, myocardial infarction and type-2 diabetes of

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human.13,14 Also, 2,4-D may influence soil microbial communities by altering the balance

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between microbial populations.3,15 In China, 2,4-D is mainly used to protect cereal crops at

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dosages ranging from 0.8 kg active ingredient (a.i.) ha-1 to 1.5 kg a.i. ha-1. The intensive use of

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2,4-D in agriculture would lead to a negative effect on the environment such as pollutions of

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water and soil.16 Although 2,4-D has a moderate persistence with relatively short half-lives in

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soils,17 it is a potential surface water and groundwater contaminant due to its relatively high water

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solubility and low sorption by soils, accentuating the importance of 2,4-D-degrading bacteria in

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water and soil.18 Therefore, biodegradation utilizing bacterial strains for efficient detoxification or

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removal of 2,4-D residues has attracted increasing attention for safe uses of 2,4-D and 3



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remediation of its pollution.

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The predominant degradation of 2,4-D is caused by microbial action in soil.19 At present, a

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variety of microorganisms capable of degrading 2,4-D have been isolated from various

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environments3 and most of them belong to the class of Proteobacteria.20 Most of 2,4-D degrading

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bacteria are composed of various genera which belong to beta-proteobacteria and

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gamma-proteobacteria.3,21 Those genera include Achromobacter, Bordetella Burkholderia,

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Cupriavidus,

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Variovorax.18,22-24 Furthermore, a few bacteria are the members of genera belonging to

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alpha-proteobacteria which contain the genera of Bradyrhizobium and Sphingomonas.24,25 These

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genera harbor tfdA-like gene (tfdA or tfdAα) or cadAB gene which could express related enzymes

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to catalyze the degradation of PAA herbicides.18 Cupriavidus necator JMP134 is a

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well-characterized 2,4-D degrading bacteria and used as a model for the study of PAA herbicides

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biodegradation.26 C. necator JMP134, formerly known as Alcaligenes eutrophus, Ralstonia

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eutropha and Waustersia eutropha,27 has a plasmid pJP4 containing the tfd genes cluster

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(tfdABCDEF genes) which can encode various enzymes for 2, 4-D biodegradation.3,26 C. necator

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JMP134 could degrade 70% of 2,4-D at 250 mg L-1 in 10 days and its degradation rate was 17.5

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mg L-1 d-1.28

Delftia,

Flavobacterium

Halomonas,

Pseudomonas,

Rhodoferax,

and

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Although numerous strains have been reported to degrade 2,4-D, it would be still valuable

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for acquiring novel and fast degradation strains as optional resources to detoxify 2,4-D in

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contamination sites. The aims of this study were to (i) isolate a highly efficient 2,4-D degrading

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bacterium, (ii) evaluate the effects of pH, temperature, substrate concentration, and inoculation

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amount on 2, 4-D biodegradation, (iii) assess its degradation capacity in soil and enzyme activity 4


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in solution, and (iv) elucidate metabolic pathway of 2, 4-D.

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2 Materials and methods

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2.1 Chemicals

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2,4-D (purity, 99.9%), 2,4-DCP (purity, 99.5%), 3,5-DCC (purity, 99.9%), 2,4-D sodium salt

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(purity, 98.5%), 2,4-D isooctyl ester (purity, 93.0%), MCPA (purity, 99.5%), MCPA isooctyl ester

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(purity, 95.0%), 2,4-DP (purity, 99.5%), 2,4-dichlorophenoxybutyric acid (2,4-DB) (purity,

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99.0 %), and 2,4,6-trichlorophenol (98.5%, purity) were purchased from Dr. Ehrenstorfer GmbH,

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Augsburg, Germany. All other chemicals and solvents were of analytical grade or HPLC grade.

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2.2 Enrichment and isolation of 2,4-D-degrading bacteria

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Soil samples used for isolating 2,4-D-degrading bacteria in this study were collected from

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the soybean field which was spayed 2,4-D over ten years in Linyi, Shandong Province, China.

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Mineral salt medium (MSM) consisted of 0.4 g L-1 of MgSO4•7H2O, 0.002 g L-1of FeSO4•7H2O,

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0.2 g L-1of K2HPO4, 0.2 g L-1 of (NH4)2SO4, 0.08g L-1 of CaSO4, 1000 mL of deionized water at

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pH 7.0.29 Ten grams of soil samples were added to a 250-mL Erlenmeyer flask containing 100

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mL MSM which was supplemented with 200 mg L-1 of 2,4-D as sole carbon and energy sources.

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The mixture was shaken and incubated at 30 °C and 150 rpm on a rotary shaker. After 7 days, 10

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mL of the culture was transferred into a fresh MSM containing 2,4-D (40 mg L-1) and incubated

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for 7 days under the same conditions described as above. Such enrichment process was repeated

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three times in a fresh MSM with the concentrations of 2,4-D increasing from 40 to 100 mg L-1,

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and then the culture was dilution-plated and spread onto sterilized MSM agar plates containing 5



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200 mg L-1 of 2, 4-D, and incubated at 30 °C in the dark. After incubation for 7 days, single

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colonies in the MSM plates containing of 2,4-D were picked aseptically, and tested for their

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degradation ability for the herbicide 2,4-D. A bacterial strain T-1 for degrading 2,4-D was

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attained from these colonies formed on the plates and used to carry out further studies.

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2.3 Identification of bacterial strain

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To

identify

the

strain

T-1,

experiments

including

morphological

observation,

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physic-biochemical tests, and 16S rRNA gene sequence analysis were performed. The strain T-1

113

grew in Luria Bertani (LB) liquid medium for 20 h at 30 °C, the cells were fixed by

114

glutaraldehyde and frozen-drying, followed by morphological observation under a scanning

115

electron microscope (SEM) (S-4800, Hitachi, Tokyo, Japan). Physic-biochemical characteristics

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were assayed by the Institute of Microbiology, Chinese Academy of Science (Beijing, China)

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according to the methods described by Dong and Cai.30 The genomic DNA of the strain T-1 was

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extracted using Tiangen Bacteria DNA Kit (Tiangen Biotech Co. Ltd., Beijing, China) according

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to the manufacturer’s instructions. The 16S rRNA gene was amplified through polymerase chain

120

reaction (PCR) using universal bacterial primers according to the method described by Liu et al.29

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16S rRNA gene sequences were sequenced by Invitrogen Biotechnology Co. Ltd., (Shanghai,

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China). Assembled 16S rRNA gene sequences of the strain T-1 was compared with that in the

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public domain through a BLAST search tool of NCBI.31 Phylogenetic tree analysis was

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constructed using the neighbor-joining method with MEGA6.0 software.32

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2.4 Inoculum preparation

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The strain T-1 was pre-cultured overnight (24 h) in LB medium at 30 °C and 150 rpm on a

127

rotary shaker. The cells were harvested after centrifugation (3380×g, 10 min), and then

128

immediately washed thrice (3 × 20 mL) with sterilized MSM. The cells of T-1 were re-suspended

129

with MSM at pH 7.0. For all degradation experiments, the density of cell was adjusted to an

130

OD600 of 0.6 (1.8× 108 cfu mL-1) determined by UV-1800 (Shimadzu Corp., Kyoto, Japan).

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2.5 Biodegradation of 2,4-D by strain T-1 in MSM

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To assess biodegradation of 2,4-D by the strain T-1, MSM was supplemented with 2,4-D as

133

the sole carbon and energy source. Each flask was inoculated with 0.5 mL suspensions of the

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strain T-1 (OD600, 0.6). All flasks were shaken at 150 rpm and 30 °C. For the control, the flask

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uninoculated T-1 was performed under the same conditions as described above. Each treatment

136

and control was carried out in triplicate. At regular time intervals, the whole cultures were

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collected, followed by addition of 20 mL of methanol. After sonication for 1 min, the mixture was

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transferred to a 50-mL volumetric flask, and finally adjusted to 50 mL with 50% aqueous

139

methanol. The solution was filtered through 0.22-µm membrane prior to the analysis of 2,4-D

140

residue.

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The effect of the initial concentration of 2,4-D in MSM on biodegradation of 2,4-D was

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tested at levels of 10-350 mg L-1. Effects of temperature on 2,4-D biodegradation were evaluated

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at 20, 25, 30, 37, and 42 °C. The solution of MSM was adjusted with buffers (0.2 mol L-1

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NaH2PO4 and Na2HPO4) at pH 5, 6, 7, 8, and 9 for the measurement of pH effect on the

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biodegradation of 2,4-D. Effects of inoculation volumes to 2,4-D biodegradation were examined 7



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with varying amounts of cell suspensions (0.2, 0.5, 1.0, 2.0, and 4.0 mL of culture, OD600 0.6).

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2.6 Biodegradation of other PAA herbicides

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An aliquot of 0.5 mL of bacteria cell suspensions (OD600, 0.6) was inoculated into 20 mL

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MSM containing each 50 mg L-1 of 2,4-D sodium salt, 2,4-D isooctyl ester, MCPA, MCPA

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isooctyl ester, 2,4-DP, 2,4-DB, or 2,4,6-trichlorophenol, followed by incubation at 30 °C in a

151

rotary shaker at 150 rpm. After 4 and 7 days, the samples were taken for determination. All

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treatments were prepared separately in triplicate and the controls were not inoculated with cell

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suspensions of T-1.

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2.7 Determination of 2,4-D and other PAA herbicides

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An aliquot of 20 mL of methanol was added to the above culture sample. The mixture was

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sonicated for 1 min, followed by adjustment of the volume to 50 mL for the analysis by a Waters

157

Alliance e2695 high performance liquid chromatograph (HPLC) equipped with a 2489 ultraviolet

158

detector (Waters Co., Milford, Massachusetts, USA). 2,4-D, 2,4-DCP, 3,5-DCC, 2,4-D sodium

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salt, 2,4-DP, MCPA, 2,4-D isooctyl ester, MCPA isooctyl ester, 2,4-DB, and 2,4,6-trichlorophenol

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were detected with HPLC. The chromatographic separation was achieved on an Agilent Eclipse

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Plus C18 column (5 µm, 4.6 mm × 250 mm) at 40 °C. The flow rate was 1.0 mL min-1 with 60%

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aqueous acetonitrile (containing 0.5% acetic acid) as the elution solution except for 2,4-D isooctyl

163

este and MCPA isooctyl ester. The sample injection volume was 20 µL. The detection wavelength

164

was 230 nm. The detection of MCPA isooctyl ester and 2,4-D isooctyl ester was conducted by the

165

same method as described above besides the proportion of elution solution of 85% aqueous 8


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166

acetonitrile (containing 0.5% acetic acid).

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2.8 Determination and identification of 2,4-D metabolites

168

An aliquot of 2.5 mL of bacteria cell suspensions (OD600, 0.6) were inoculated in 100 mL of

169

MSM containing 2,4-D (100 mg L-1). After incubation for 0-36 h at 30 °C and 150 rpm, the

170

cultures were extracted according to the following methods: (1) At 4, 8, 10, 12, 16, 20, 24, and 36

171

h, an aliquot of 2 mL of culture samples were filtered through a 0.22-µm membrane and analyzed

172

on a HPLC and a Waters AcquityTM ultra-performance liquid chromatograph coupled to XEVO

173

triple quad mass spectrometer (UPLC-MS/MS, Waters). (2) After incubation for 8 and 16 h, the

174

whole culture was frozen and dried by a FD5-4 freezer dryer (SIM International Group Co., Ltd.,

175

Los Angeles, California, USA), and then was dissolved in 5 mL of ethyl acetate or methyl alcohol.

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The dissolved sample was filtered through a 0.22-µm membrane prior to further analysis with an

177

Agilent 7890B gas chromatograph equipped with an Agilent 7000C triple quad mass

178

spectrometer (GC-MS/MS, Agilent Technology, Santa Clara, California, USA) and an Agilent

179

1290 infinity II UPLC coupled to 6545 quadrupole time-of-flight mass spectrometer

180

(UPLC-QTOFMS, Agilent Technology).

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2,4-D metabolites were detected with UPLC-MS/MS. An analytical column (Waters BEH

182

C18, 1.7 µm, 2.1 mm × 100 mm) was used for chromatographic separation with a mobile phase of

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water containing 0.1% formic acid (A) and methanol (B) at a constant flow rate of 0.3 mL min-1.

184

The gradient elution program was isocratic 10% B for 0.5 min, from 10% to 70% of B over 7.5 min,

185

from 70% to 10% of B over 0.2 min, and 10% for 1.8 min prior to the next injection. The mass

186

spectrometer was operated at electrospray ionization mode and the scan ranged from m/z 100 to

187

300. Nitrogen was used as the desolvation and cone gases with 800 and 50 L h-1 of flow rates,

188

respectively. Desolvation (400 °C) and ion source (150 °C) were kept at constant temperatures.

189

The voltage was 1000 V and 21 V of capillary and cone, respectively. 9



190

The lyophilized samples were analyzed with GC-MS/MS. A HP-5 MS silica capillary

191

column (0.25 µm, 30 m × 0.25 mm) was selected as the separation column. Helium was used as

192

carrier gas at a flow rate of 1.2 mL min-1. The oven temperature started at 60 °C for 0.5 min, then

193

increased to 200 °C at a rate of 10 °C min-1 and held for 1 min, and then raised to 280 °C at a rate

194

of 15 °C min-1 and held for 10 min. Interface (280 °C), injector (270 °C), and ion source (280 °C)

195

were kept at constant temperatures. The mass spectrometer was operated with electron ionization

196

(EI) source. Electron ionization energy was set at 70 eV. The MS detector was scanned from m/z

197

50 to 600.

198

The lyophilized samples were also analyzed with UPLC-QTOFMS. The analytical condition

199

of UPLC-QTOFMS was the same as that of UPLC-MS/MS except for some MS parameters. The

200

mass spectrometer was operated with electrospray ionization (ESI) source at negative ion model

201

with voltage of 3.5 KV. Nitrogen was used as the sheath gas at a flow rate of 11 L min-1 and

202

temperature was set at 350 °C. The fragmentor and skimmer voltages were set to 100 V and 65 V,

203

respectively. The TOF data were collected between m/z 50-350.

204

2.9 Biodegradation of the metabolites 2,4-DCP and 3,5-DCC

205

An aliquot of 0.5 mL of bacterial cell suspensions (OD600, 0.6) were inoculated in 20 mL of

206

MSM containing 10 mg L-1 of 2,4-D, 2,4-DCP, 3,5-DCC, or their mixture. All flasks were

207

incubated in a rotary shaker at 30 °C and 150 rpm in the dark. At certain time intervals, the whole

208

culture was collected for determination of the concentration of 2,4-D, 2,4-DCP or 3,5-DCC. Each

209

treatment was carried out in triplicate, and the control treatment without T-1 was performed under

210

the same conditions.

211

2.10 Biodegradation of 2,4-D in soil

212

Soil used for degradation assays was collected from a field located at Changfeng County, 10


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213

Hefei, China. Surface soil taken from the top layer (0-15 cm) was mixed thoroughly, and then

214

passed through 2-mm sieve to remove stones and debris. The soil was classified as silty clay loam,

215

and its properties were as follows: sand, 25.4%; silt, 51.2%; clay, 24.1%; organic matter content,

216

4.19%; water holding capacity, 30.4%; cationic exchange capacity, 18.1 cmol kg-1; total nitrogen,

217

0.18%; and pH 7.4. Soil samples (0.5 kg dry-weight equivalent) were treated with methanol

218

solution of 2,4-D to obtain the final concentrations of 10 mg kg-1 and 50 mg kg-1 of dry soil and left

219

for 1 hour on a laminar flow bench for methanol evaporation. The soil samples were inoculated

220

with the strain T-1 preparation to give an initial inoculum density of 9.0×106 cfu g-1 soil, mixed

221

thoroughly with a plastic spoon, and passed through a mesh (2 mm) to distribute 2,4-D and T-1

222

bacteria. The soil samples receiving the same amount of 2,4-D, but without inoculums were used as

223

the controls. Soil water content was adjusted to 60% of water holding content and maintained

224

constantly by periodic addition of sterile distilled water. Soil samples were transferred to a 1-L

225

conical flask with stopper and incubated at 30 °C in the dark. Each treatment was performed in

226

triplicate. After incubation for 0, 6, 12, 18, 24, 36, 48, 60, 72, and 96 h, 20 g of soil samples from

227

each treatment were taken for analysis of residual 2,4-D.

228

For determination of 2,4-D concentration in soil, soil samples (5 g) were extracted with 40 mL

229

of 0.04 M NaOH solution for 50 min at 150 rpm on a rotary shaker and sonicated for 10 min. After

230

centrifugation at 3380×g for 5 min, 15 mL of the supernatant was transferred to a 50-mL

231

centrifuge tube and adjusted to pH 3 with 85% phosphoric acid. Subsequently 20 mL of ethyl

232

acetate and 5 g of NaCl were added to the centrifuge tube. The mixtures were sonicated for 20

233

min and centrifuged at 3380×g for 5 min. The ethyl acetate phase was collected, concentrated to

234

dryness, and finally dissolved in 5 mL of methanol-water (1:1, V/V). The mean recoveries of 11



235

fortified 2,4-D in soil at 0.05, 1, 10, and 50 mg kg-1 varied from 94%% to 96% with a relative

236

standard deviation (RSD) ≤ 7%. These data indicated that the method was satisfactory for the

237

analysis of 2,4-D in soil.

238

2.11 Degradation of 2,4-D by crude enzyme

239

For the preparation of crude enzyme, the cells of T-1 pre-grown for 24 h in LB medium were

240

exposed to 2,4-D at a concentration of 200 mg L-1 in MSM for 8 h at 30 °C for enzyme induction.

241

The cells were collected by centrifugation (3380×g, 10 min) at 4 °C and then immediately washed

242

thrice (3 × 20 mL) with sterilized phosphate buffer solution (PBS, 50 mM NaH2PO4-Na2HPO4,

243

pH 7.0). The wet cells (1 g) were re-suspended in 15 mL PBS. Extraction of extracellular and

244

intracellular crude enzymes of T-1 cells were referred to the method reported by Liu et al.29 The

245

cell debris containing membrane-bound enzymes were re-suspended in 15 mL of PBS and kept at

246

4 °C for later use. The activities of different crude enzymes were determined by analyzing the

247

amount of 2,4-D degraded. One unit of enzyme activity was defined as the amount of enzyme

248

resulting in the degradation of 1 µmol 2,4-D per min at 30 °C.

249

To determine effects of α-ketoglutarate (α-KG) and Fe2+ on enzyme activity, 0.5 mL of crude

250

enzymes (extracellular, intracellular or membrane-bound enzymes) were added to 2.5 mL of PBS

251

containing 2,4-D and the concentration of 2,4-D in mixed solution was 0.226 mM. The enzyme

252

activities of the mixed solution were examined in the presence of 1 mM α-KG, or 1mM Fe2+, or

253

with both compounds in comparison with no α-KG and Fe2+ as the control. Each treatment was

254

performed in triplicate.

255

After incubation for 5 min at 30 °C, the enzyme reaction was stopped by the addition of 0.2 12


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256

mL of hydrochloric solution (1 mM). Methanol (3 mL) was added to the culture sample, followed

257

by sonication for 1 min. 2,4-D residues were determined according to the method described

258

above.

259

2.12 Calculation and statistical analysis

260 261

The degradation percentage and degradation rate were calculated from the following equation (1) and (2):

262

X =

263

V =

× 100% (1)

(2)

264

where X (%) is the degradation percentage of 2,4-D; V (mg L-1 h-1) is degradation rate of

265

2,4-D; Cck and Ct are control concentration and treatment residual concentration (mg L-1) of 2,4-D,

266

respectively; t is the culture time.

267

All treatments were performed in triplicate and arithmetic averages were taken throughout

268

the data analysis and calculation. The data were calculated using Excel (Microsoft 2010, USA)

269

and then analyzed with SPSS 19.0 software package (SPSS Inc., USA) and Origin 8.0 software

270

(OriginLab Co., USA). The statistical significance was determined at p

Rapid Biodegradation of the Herbicide 2,4-Dichlorophenoxyacetic

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